This grant focuses on translational research to advance neuroimaging techniques that will enhance the potential of human neural stem cell (hNSC) implantation to treat neonatal hypoxic- ischemic brain injury (HII). Our goal is to use advanced magnetic resonance imaging (MRI) in a standard rat pup model of unilateral middle cerebral artery occlusion with hypoxia (Rice- Vannucci, RVM) to: (1) monitor non-invasively (as might be done clinically) hNSC migration, proliferation, location and as an outcome biomarker; (2) use imaging to tailor optimal implantation (site, dose, and timing); and (3) develop candidate selection criteria based on imaging model injury severity. Using an 11.7T MRI, we have developed: (1) a automated 3D MRI volumetric method to measure total and regional HII volumes; (2) a MRI based Rat Pup Scoring System (RPSS); (3) a 3-tiered model of HII severity (mild, moderate, severe) based on 3D HI volumes and the RPSS; and (4) 3 quantifiable neuroimaging parameters (migration, proliferation and final location) using Feridex-labeled hNSCs that can be compared with 4 quantifiable histological, immunohistochemistry and neurophysiological parameters of hNSC fate (integration, differentiation, connectivity, and survival). These data will be evaluated at 3 months against an extensive battery of behavioral testing. Aim 1a will examine whether neuroimaging parameters correlate with parameters of hNSC fate. Aim 1b will examine if iron labeling adversely affects NSCs or causes additional tissue injury. Aim 2 will assess the efficacy of neuroimaging to determine site, dose, and timing of implantation for optimal structural and metabolic recovery. Aim 3 will use neuroimaging to examine whether the ability of hNSCs to improve structural, metabolic and behavioral outcomes depends upon the severity of the initial HII using the RVM and a new model of bilateral injury (bilateral carotid occlusion with hypoxia; BCAO-H). This grant addresses many of the important technical and biological issues that must be considered in order to improve the chance for success of NSC therapy. By using advanced imaging methods to determine HII severity, injury location and injury volumes, we have developed a unique and objective approach to evaluate efficacy of hNSC implantation. hNSC treatment is an extremely promising approach to treat HII, but we have an obligation to provide the scientific foundation in a careful, objective, logical and safe manner before embarking on clinical trials in newborns. PUBLIC HEALTH RELEVANCE: This grant focuses on translational research to advance neuroimaging techniques that will enhance the potential of human neural stem cell (hNSC) implantation to treat neonatal hypoxic- ischemic brain injury (HII). Our goals are to use advanced magnetic resonance imaging (MRI) techniques as an outcome measure and to: (1) monitor non-invasively (as might be done clinically) hNSC migration, proliferation and location; (2) optimize hNSC implantation site, dose, and timing; and (3) identify potential therapeutic candidates based on early MRI measures of injury severity. Using an 11.7T MRI in two models ischemia and hypoxia we have developed automated 3D MRI volumetric methods to measure total and regional HII volumes and NSC migration. This proposal will have a significant impact on treating neonatal HII with hNSCs. Development of automated MRI methods that objectively quantify injury, confirm precision of implantation, monitor hNSC activity, and quantify brain volumetric recovery is critical for translational research as a prelude to trials in higher order species and then clinical trials.